Editing Synthesis Peptide-PEI (section)

== TAT-PEG-PEI<br>  ==

Tat GRKKKRRQRC<br> 

α-vinyl sulfone-? -N-hydroxysuccinimide ester poly(ethylene<br>glycol) (NHS-PEG-VS) was purchased from Nektar Therapeutics (Huntsville, USA). The 25<br>kDa branched polyethylenimine (BPEI)<br> 

Activation of BPEI<br>19.2 mg (565 μmol) of bifunctional PEG (3.4 kDa), containing both an α-vinyl sulfone<br>and an ω-N-hydroxysuccinimide ester group, was weighed into a glass flask 4.293 μl of a<br>BPEI solution (corresponding to 12.15 mg/0.486 μmol BPEI; 282.4 μmol total amines) in<br>0.1M borate buffer at pH 5.5 were added and stirred. The activation reaction was carried out<br>for 4 hours at room temperature, followed by pH adjustment to 7 with 1N chloride acid and<br>additionally incubated for 2 hours at room temperature.<br>Coupling of the oligopeptide onto activated PEG-PEI<br>For coupling, 2.98 mg (2.26 μmol) of the oligopeptide TAT dissolved in 866 μl pure<br>water were added to the activated PEG-PEI yielding a theoretical degree of substitution of<br>approximately 1% based upon amine functions. The mixture was stirred for additional 2 hours<br>at room temperature.<br> 

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Coupling of the oligopeptide onto activated PEG-PEI<br>For coupling, 2.98 mg (2.26 μmol) of the oligopeptide TAT dissolved in 866 μl pure<br>water were added to the activated PEG-PEI yielding a theoretical degree of substitution of<br>approximately 1% based upon amine functions. The mixture was stirred for additional 2 hours<br>at room temperature.<br>Purification of TAT-PEG-PEI<br>Removal of unreacted PEG and low molecular weight residues was performed with an<br>ultrafiltration cell (Amicon, Bedford, USA) equipped with a 10 kDa molecular weight cut-off<br>membrane (Millipore, Bedford, USA) and 0.1 M borate buffer at pH 7.5 as eluent.<br>Amine concentration of the conjugate was determined by a recently described copper<br>assay [27].<br> 

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The (polyethylene glycol)-graft PEIs<br>(PEGPEI), were synthesized as previously described [28]. Briefly, PEGPEIs were obtained by<br>activating PEG-monomethyl ether with a hexamethylene diisocyanate. The coupling of PEG<br>to the amino groups of BPEI was carried out in anhydrous chloroform at 60 °C, which led to a<br>the PEI copolymers: (PEG5k) 2 PEI containing 2 PEG 5 kDa molecules per molecule of BPEI<br>and (PEG550) 35 PEI containing 35 550 Da molecules per molecule of BPEI.<br> 

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Briefly, PEGPEI was obtained by activating PEG-monomethyl<br>ether (5 kDa) with a hexamethylene diisocyanate. The coupling of PEG to the amino groups<br>of BPEI was carried out in anhydrous chloroform at 60 °C, which led to a PEI copolymer<br>containing 2 PEG 5 kDa molecules per molecule of BPEI.<br> 

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